An approach to crystallizing proteins by synthetic symmetrization

Abstract

Previous studies of symmetry preferences in protein crystals suggest that symmetric proteins, such as homodimers, might crystallize more readily on average than asymmetric, monomeric proteins. Proteins that are naturally monomeric can be made homodimeric artificially by forming disulfide bonds between individual cysteine residues introduced by mutagenesis. Furthermore, by creating a variety of single-cysteine mutants, a series of distinct synthetic dimers can be generated for a given protein of interest, with each expected to gain advantage from its added symmetry and to exhibit a crystallization behavior distinct from the other constructs. This strategy was tested on phage T4 lysozyme, a protein whose crystallization as a monomer has been studied exhaustively. Experiments on three single-cysteine mutants, each prepared in dimeric form, yielded numerous novel crystal forms that cannot be realized by monomeric lysozyme. Six new crystal forms have been characterized. The results suggest that synthetic symmetrization may be a useful approach for enlarging the search space for crystallizing proteins.

Fig. 1.

Diagram illustrating the idea of symmetrization combined with cysteine mutagenesis. The protein molecule is represented by the figure “5.” Cysteine residues are yellow. Each construct would be expected to present independent crystallization opportunities. Dimerization is achieved by direct disulfide bond formation. Trimerization would require a trivalent thiol-specific cross-linking reagent. Trimerization experiments are not discussed here.

Fig. 2.

The sites for generation of three single-cysteine lysozyme mutants used in synthetic dimerization and crystallization experiments. The native cysteine residues (C54 and C97) that had been removed by earlier mutagenesis (30) are shown in red. The individual sites for new cysteine residues (S44, D72, and V131) are shown in yellow.

Fig. 3.

Six new crystal forms of synthetic T4 lysozyme dimers. (A) Crystal packing diagrams. In each case, red dots indicate the location of the single engineered disulfide bond. The annotation below each image indicates the site of the cysteine residue, the crystallization conditions (see Methods), and the crystal space group (see Table 1). (B) Electron density maps confirming the presence of disulfide bonds in the synthetic dimers: crystal form i (Left) V131C, crystal form ii (Center) S44C, and crystal form v (Right) D72C. 2F_obs − F_calc maps are shown, contoured at 1.0 standard deviations, showing that the disulfide bonds (yellow) are present in all cases.

Fig. 4.

An analysis of crystal space group preferences in the PDB. (A) Comparison of the results for monomeric and dimeric proteins. (B) An illustration of the calculation of increased crystallization likelihood for dimeric proteins. The height indicated by “expected” is the number of dimer crystals that would be expected if dimeric proteins gained no crystallization advantage from their internal symmetry. The ratio of observed to expected heights is 1.46.